Protein N-terminal methyltransferases (NTMTs) are a relatively new type of protein methyltransferase that catalyze the methylation of alpha-N-terminal amines of proteins starting with an X-P-K/R motif. The alpha-N- terminal methylation plays an essential role in regulating cell mitosis, chromatin interactions, and DNA repair. Its level is increased as response of cellular stress, aging, and developmental processes. Aberrant expression of NTMTs has been observed in various human diseases. However, the biological impact of protein alpha-N- terminal methylation and the molecular basis of NTMT catalysis are poorly defined. Our long-term research goal is to elucidate the biochemical pathways mediated by NTMTs that contribute to the pathogenesis of cancer and developmental defects, and to develop potent and specific NTMT inhibitors. The objective of this research to study mechanism, regulation, and recognition of protein alpha-N-terminal methylation through exploring and applying new chemical biology approaches. Three specific aims will be pursued: Aim 1. Elucidate the molecular and structural basis for substrate and product specificity of NTMTs. We will apply mutational and crystallographic analyses to understand how NTMT1 and 2 recognize their substrates and produce specific methylated products. In addition, we propose to identify a bioorthogonal pair of mutant NTMT1 and SAM analog and use it to obtain a comprehensive profile of physiological substrates for NTMT1. We will carry out biochemical assays and cellular methylation assays to confirm the relevance of identified substrates in cells. Aim 2. Develop potent and specific inhibitors for NTMTs. We will build on our preliminary data to complete synthesis and evaluation of a series of bisubstrate analogs for NTMTs, which will be used as probes to characterize the structural and functional differences of NTMT1 and 2, to investigate NTMT inhibition in cells in comparison with the effects of NTMT1 knockdown. We will extend our crystal structure database of NTMT ternary complexes and to screen for small molecule inhibitors for NTMTs. Aim 3. Identify readers and erasers for protein alpha-N-terminal methylation. There is no information on how this N-terminal methylation is `read' by interacting molecular partners and whether it is static or dynamic (`erasable'). We propose to prepare photoaffinity labeling probes as baits to identify interacting partners of protein alpha-N-terminal methylation in cell extracts. Identified interacting partners will be validated through binding studies and immunoprecipitation experiments. Bioinformatics analysis will be done to develop hypotheses for the biological functions of these interacting partners. Taken together, we believe that this research effort has the great potential to provide a clearer understanding of mechanisms and inhibition of NTMTs, and shed lights on the biological impact of protein N-terminal methylation. Accomplishment of the proposed work will also provide new chemical tools for both basic NTMT biology research and facilitate the development of novel therapeutic approaches to target NTMT1 and NTMT1-involved pathways.